PROJECT SUMMARY The primary goal of this R21 proposal is to characterize and systematically identify novel host serine protease inhibitors (SERPINs) that restrict replication of three clinically important respiratory viruses. Although respiratory viruses pose a major health burden, there is a lack of effective treatment options. Understanding the mechanisms by which host factors inhibit these viral infections may lead to novel antiviral strategies. The project is founded on our recent discovery that one SERPIN family member, SERPINE1, inhibits influenza A virus by targeting airway proteases that the virus requires for glycoprotein maturation. This study opened the door for a novel concept in innate immunity: that SERPINs may constitute a previously unappreciated class of antiviral factors. The main technical innovation of this proposal is the use of stratified polarized airway epithelium cultures (HAEC) inducibly expressing individual SERPINs from a recently assembled library. These HAEC display the entire arsenal of endogenous intra- and extracellular airway proteases, a number of which play important roles in respiratory virus life cycles. This proteolytic environment allows for a more unbiased discovery of SERPINs that inhibit endogenous airway proteases and thereby may restrict replication of specific respiratory viruses. We are combining this 3D tissue culture technology with confocal high-content imaging, directly on the polarized cultures. Completion of our aims may provide a novel facet of the innate immune response: modulating proteolytic tissue environments to inhibit viruses.